Technical Abstract:
Norovirus (NV), hepatitis A virus (HAV), and other virus transmission by molluscan shellfish is a significant issue. Research at the ARS-Dover DE laboratory has led to the development of improved methods for detecting these viruses. To identify pathogenic viruses within mollusks, a rapid highly-sensitive four-step virus RNA extraction method, using glycine buffer, polyethylene glycol, tri-reagent, and poly dT magnetic beads, has been developed. This method, termed the GPTT procedure, facilitates reverse transcription-polymerase chain reaction (RT-PCR) detection of HAV and NV. This procedure detects less than one plaque-forming unit (PFU) of HAV and approximately 22.4 RT-PCR units of NV. It has been successfully adapted to detect both HAV and NV within imported clams implicated in an outbreak of viral gastroenteritis within the United States. This method has also been adopted as a standard method by the Food Directorate of the Canadian Government, and was used by their laboratories to identify NV within outbreak associated with oysters. The GPTT method has been used to detect a number of picornaviruses including Aichi virus, coxsackieviruses A9 and B5, human parechovirus-1, as well as other positive-strand RNA viruses such as hepatitis E and feline calicivirus (FCV).
Using the GPTT method, the ability of HAV to persist within Eastern oysters (Crassostrea virginica) was investigated. Viral RNA was detected by RT-PCR 6 weeks after a 16 h exposure to 90,000 PFU (180 PFU/ml seawater) of HAV. Assays for infectious virus in oysters that received a daily feeding of phytoplankton, demonstrated recovery of 3800, 650, and 500 PFU of HAV, 1-, 2-, and 3-weeks post-contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4-, 5-, or 6-weeks post-contamination. These results support the position that shellfish depuration is insufficient for complete removal of infectious virus. Extended relay times, in excess of 4 weeks, may be required to produce virologically-safe shellfish.